Cytoplasmic regulatory factors appear to play an important role in the early development of fertilized eggs. We feel they also have an important role in differentiation and our studies attempt to elucidate this role using cultured mammalian cells as a model system. We have used cybrids for study of the control of differentiated functions by cytoplasmic regulatory factors. Extinction of hemoglobin induction and activation of phenylalanine hydroxylase (PH) in mouse erythroleukemia (MEL) cybrids suggest the existence of self-perpetuating negative and positive cytoplasmic regulatory factors respectively. Isoelectirc focusing and immunotitration studies indicate that MEL cybrids express a mouse form of PH. Using physical and chemical agents to inactivate proteins and nucleic acids, we have concluded that the cytoplasmic regulatory factor for PH gene is perhaps RNA in nature. To confirm that RNA is involved in the cytoplasmic regulation of PH gene, MEL cells were selected in tyrosine-free medium, after transfection with RNA, isolated from cells expressing PH, using the calcium phosphate precipitation technique. Chromatofocusing and immunotitration studies indicate that RNA-transfected MEL cells express the mouse form of enzyme. Our long range plan is to elucidate the mechanism of regulation of a differentiated function by cytoplasmic RNA regulators, using biochemical genetics approaches and cloning technology.